# US 10,501,743 B2 · Methods and compositions for selection of functional oligonucleotides

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- Number: US 10,501,743 B2
- Document ID: US10501743B2
- Status: Granted
- Kind: B2
- Application number: 16/165,267
- Filed: 2018-10-19
- Published: 2019-02-07
- Granted: 2019-12-10
- Earliest claimed: 2017-03-30
- Applicants: Augmanity Nano LTD
- Inventors: Ido Bachelet; Noam Mamet; Itai Rusinek; Gil Harari; Anastasia Shapiro; Yaniv Amir; Erez Lavi; Almogit Abu-Horowitz
- Web page: https://patentgrep.com/patent/US10501743B2

## CPC

- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- C12N 15/1048: {SELEX}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12N 2310/16: Aptamers
- C12N 2320/13: in a process of directed evolution, e.g. SELEX, acquiring a new function
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present disclosure describes compositions and methods for rapid selection of both binding and functional oligonucleotides (DNA, RNA, or any natural or synthetic analog of these). In certain embodiments, provided herein are flow cells (e.g., flow cells for an Illumina sequencing instrument or a Polonator sequencing instrument) comprising within its flow chamber a plurality of immobilized aptamer clusters (e.g., from an aptamer library described herein) and, optionally, one or more target cells (e.g., cancer cells, immune cells, etc.) and/or a detectable indicator of cellular function (e.g., a fluorescent indicator of apoptosis, cell proliferation, gene or protein expression, etc.). In certain embodiments, provided herein are methods of using such an aptamer cluster-containing flow cell to identify functional aptamers from an aptamer library (e.g., in a sequencing instrument, such as an Illumina sequencing instrument).

## Claims

### Claim 1

A method for identifying one or more aptamers that interact with a target cell, the method comprising:
(i) contacting a plurality of aptamer clusters immobilized on a surface with a target cell wherein at least 102 distinct aptamer clusters are immobilized on the surface, and wherein each aptamer cluster:
(1) is of a known sequence; and
(2) is at a known position on the surface;
(ii) determining positions on the surface at which the target cell is retained; and
(iii) identifying the aptamer clusters immobilized on the surface at the positions at which the target cell is retained as the immobilized aptamer clusters that interact with the target cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the surface is a surface of a flow cell.

### Claim 3 (depends on 1)

The method of claim 1, further comprising the steps of:
(a) immobilizing a plurality of aptamers from an aptamer library on a surface; and
(b) amplifying the plurality of immobilized aptamers locally on the surface to form the plurality of immobilized aptamer clusters.

### Claim 4 (depends on 3)

The method of claim 3, wherein the amplification is conducted via bridge PCR amplification or rolling circle amplification.

### Claim 5 (depends on 4)

The method of claim 4, wherein the method further comprises removing the complementary strands from the immobilized aptamer clusters to provide single stranded immobilized aptamer clusters.

### Claim 6 (depends on 1)

The method of claim 1, further comprising an aptamer folding step, wherein the aptamer folding step comprises raising and then lowering the temperature of the aptamer clusters.

### Claim 7 (depends on 1)

The method of claim 1, further comprising an aptamer folding step, wherein the aptamer folding step comprises adding a denaturing agent to the aptamer clusters and then removing the denaturing agent from the aptamer clusters.

### Claim 8 (depends on 1)

The method of claim 1, wherein the immobilized aptamer clusters are sequenced before step (i).

### Claim 9 (depends on 8)

The method of claim 8, wherein the sequencing is conducted via Illumina sequencing or Polonator sequencing.

### Claim 10 (depends on 1)

The method of claim 1, wherein at least 108 distinct aptamers are immobilized on the surface and each aptamer cluster comprises 103 to 106 copies of an aptamer.

### Claim 11 (depends on 1)

The method of claim 1, wherein the target cell is labeled with a reporter of cell function.

### Claim 12 (depends on 11)

The method of claim 11, wherein the cell function is apoptosis, caspase-3/7 activity, proliferation, gene expression, or cytokine expression.

### Claim 13 (depends on 11)

The method of claim 11, comprising the step of identifying the immobilized aptamer clusters that mediate a cell function in the target cell by detecting the reporter of cell function.

### Claim 14 (depends on 13)

The method of claim 13, wherein the cell function is apoptosis.

### Claim 15 (depends on 1)

The method of claim 1, wherein the positions on the surface at which the target molecule is retained are determined by fluorescent microscopy.

### Claim 16 (depends on 1)

The method of claim 1, wherein the aptamers in the aptamer cluster comprise nucleic acid adapters binding the aptamers to the surface.

### Claim 17 (depends on 16)

The method of claim 16, wherein a sequence complementary to the nucleic acid adapter sequence is hybridized to the adapter prior to step (i).

### Claim 18

A method for identifying one or more aptamers that mediate a functional effect on a target cell, the method comprising:
(i) contacting a plurality of aptamer clusters immobilized on a surface with a target cell labeled with a reporter of cell function, wherein at least 102 distinct aptamer clusters are immobilized on the surface, and wherein each aptamer cluster:
(1) is of a known sequence; and
(2) is at a known position on the surface;
(ii) determining positions on the surface at which the target cell undergoes the functional effect by detecting the reporter of cell function; and
(iii) identifying the aptamer clusters immobilized on the surface at the positions at which the target cell undergoes the functional effect as the immobilized aptamer clusters that mediate the functional effect on the target cell.

### Claim 19 (depends on 18)

The method of claim 18, wherein the surface is a surface of a flow cell.

### Claim 20 (depends on 18)

The method of claim 18, wherein the reporter of cell function is a fluorescent reporter of cell function.

### Claim 21 (depends on 18)

The method of claim 18, further comprising the steps of:
(a) immobilizing a plurality of aptamers from an aptamer library on a surface; and
(b) amplifying the plurality of immobilized aptamers locally on the surface to form the plurality of immobilized aptamer clusters.

### Claim 22 (depends on 21)

The method of claim 21, wherein the amplification is conducted via bridge PCR amplification or rolling circle amplification.

### Claim 23 (depends on 18)

The method of claim 18, wherein the immobilized aptamer clusters are sequenced before step (i).

### Claim 24 (depends on 18)

The method of claim 18, wherein at least 108 distinct aptamers are immobilized on the surface and each aptamer cluster comprises 103 to 106 copies of an aptamer.

### Claim 25 (depends on 20)

The method of claim 20, wherein the positions on the surface at which the target cell undergoes the functional effect are determined by fluorescent microscopy.

### Claim 26 (depends on 18)

The method of claim 18, wherein the functional effect is cell death, caspase-3/7 activity, proliferation, gene expression, or cytokine expression.

### Claim 27 (depends on 18)

The method of claim 18, wherein the functional effect is cell death and the reporter of cell function is a cell death reporter.

### Claim 28 (depends on 27)

The method of claim 27, wherein the cell death reporter is 7-AAD or Annexin V.

### Claim 29 (depends on 27)

The method of claim 27, wherein the cell death is apoptosis and the cell death reporter is a fluorescent indicator of apoptosis.

### Claim 30 (depends on 29)

The method of claim 29, wherein the fluorescent indicator of apoptosis is Annexin V or green fluorescent caspase 3/7 activity reporter dye.